Methods for treatment or diagnosis of disease or disorders associated with an APB domain

ABSTRACT

The present invention concerns methods for diagnosis and treatment of diseases or disorders characterized by abnormal cellular signal transduction involving a newly identified region, herein termed the “APB domain.” APB domain binding between proteins is believed to play an important role in signal transduction pathways and, thereby, influence cellular events. Thus, APB mediated activity plays a role in signal transduction pathways and agents modulating APB mediated activity can be used to treat diseases or disorders involving proteins containing APB domains.

FIELD OF THE INVENTION

The present invention relates generally to the fields of chemistry,biology, and medicine and more specifically to the diagnosis andtreatment of various diseases or disorders.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art none of which is admittedto be prior art to the invention.

Receptor tyrosine kinases belong to a family of transmembrane proteinsand have been implicated in cellular signaling pathways. The predominantbiological activity of some receptor tyrosine kinases is the stimulationof cell growth and proliferation, while other receptor tyrosine kinasesare involved in arresting growth and promoting differentiation. In someinstances, a single tyrosine kinase can inhibit, or stimulate, cellproliferation depending on the cellular environment in which it isexpressed. (Schlessinger, J. and Ullrich, A., Neuron, 9(3):383-391,1992.)

Receptor tyrosine kinases are composed of at least three domains: anextracellular ligand binding domain, a transmembrane domain and acytoplasmic catalytic domain that can phosphorylate tyrosine residues.Ligand binding to membrane-bound receptors induces the formation ofreceptor dimers and allosteric changes that activate the intracellularkinase domains and result in the self-phosphorylation(autophosphorylation and/or transphosphorylation) of the receptor ontyrosine residues. Individual phosphotyrosine residues of thecytoplasmic domains of receptors may serve as specific binding sitesthat interact with a host of cytoplasmic signalling molecules, therebyactivating various signal transduction pathways (Ullrich, A. andSchlessinger, J., 1990, Cell 61:203-212).

The intracellular, cytoplasmic, non-receptor protein tyrosine kinases donot contain a hydrophobic transmembrane domain and share non-catalyticdomains in addition to sharing their catalytic kinase domains. Suchnon-catalytic domains include the SH2 domains (SRC homology domain 2;Sadowski, I. et al., Mol. Cell. Biol. 6:4396-4408; Koch, C. A. et al.,1991, Science 252:668-674) and SH3 domains (SRC homology domain 3;Mayer, B. J. et al., 1988, Nature 332:269-272). Such non-catalyticdomains are also thought to include the PH domain (Musacchio et al.,1993, TIBS 18:342-348). The non-catalytic domains are thought to beimportant in the regulation of protein-protein interactions duringsignal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).

A central feature of signal transduction (for reviews, see Posada, J.and Cooper, J. A., 1992, Mol. Biol. Cell 3:583-392; Hardie, D. G., 1990,Symp. Soc. Exp. Biol. 44:241-255), is the reversible phosphorylation ofcertain proteins. Receptor phosphorylation stimulates a physicalassociation of the activated receptor with target molecules. Some of thetarget molecules are in turn phosphorylated. Such phosphorylationtransmits a signal to the cytoplasm. Other target molecules are notphosphorylated, but assist in signal transmission by acting as adaptermolecules for secondary signal transducer proteins. For example,receptor phosphorylation and the subsequent allosteric changes in thereceptor recruit the Grb-2/SOS complex to the catalytic domain of thereceptor where its proximity to the membrane allows it to activate rasPawson, T. and Schlessinger, J., Current Biol. 13:434, 1993.

The secondary signal transducer molecules generated by activatedreceptors result in a signal cascade that regulates cell functions suchas cell division or differentiation. Reviews describing intracellularsignal transduction include Aaronson, S. A., Science, 254:1146-1153,1991; Schlessinger, J. Trends Biochem. Sci., 13:443-447, 1988; andUllrich, A., and Schlessinger, J., Cell, 61:203-212, 1990.

Abnormalities in signal transduction pathways can lead to variousdiseases in at least three different ways: (1) under-activity (2)mutation, and (3) over-activity. An example of under-activity isobserved in some forms of diabetes. Examples of mutation include therole of BCR-ABL in chronic myelogenous leukemia and acute lymphocyticleukemia. Pendergrast et al., 1993, Cell 75:175-185.

Over-activity of certain protein tyrosine kinases has been shown tosubvert normal growth control pathways and lead to oncogenesis (reviewedin Hunter, T., 1991, Cell 64:249-270). An example of a protein that mayparticipate in the aberrant growth of breast cancer cells is HER2, alsoknown as c-erbB-2 (Coussens et al., 1985 Science 230:1132-1139; Yamamotoet al., 1986, Nature, 319:521-527). This receptor was also isolated asthe rat oncogene neu, an oncogene responsible for chemically induced ratglioblastomas (Padhy et al., 1982 Cell, 28:865-871; Schechter et al.,1984 Nature 312:513-516; Bargmann et al., 1986, Nature, 319:226-230).HER2/erbB-2 is known to be amplified and over-expressed in about 25% ofhuman breast cancers (Slamon et al., 1987 Science 235:177-182; Slamon etal., 1989 Science 244:707-712).

SUMMARY OF THE INVENTION

The present invention concerns methods for diagnosis and treatment ofdiseases or disorders characterized by abnormal cellular signaltransduction involving a newly identified region, herein termed the “APBdomain.” APB domain binding between proteins is believed to play animportant role in signal transduction pathways and, thereby, influencecellular events. Thus, APB mediated activity plays a role in signaltransduction pathways and agents modulating APB mediated activity can beused to treat diseases or disorders involving proteins containing APBdomains.

For example, phosphorylated receptor tyrosine kinases such as EGF, TrkA,and HER-2 have an APB recognition region able to bind to an APB domainpresent in an adapter molecule such as Shc. The bound adapter moleculeis in turn phosphorylated and can interact with other molecules. Thus,signal transduction starting with phosphorylation of a receptor tyrosinekinase is in part transmitted through APB binding and modulating APBmediated activity will increase or decrease such signal transduction.

An example of an APB domain is that present in the N-terminal aminoacids 1-163 of 46 Kd Shc (p46^(shc)), and amino acids 46-209 of 52 KdShc (p52^(shc)) (see Example infra). Other proteins having an APB domainare believed to exist or can be produced synthetically. Such other APBdomains have at least 20% sequence identity or at least 30% sequencesimilarity with the APB domain present in Shc (i.e., amino acid 46-209of p52^(shc))

The APB domain can bind to a protein amino acid region (i.e. an “APBrecognition” region) present in a protein binding partner. A broad rangeof sequences may be capable of interacting with an APB domain. The APBdomain is believed to bind to a recognition region of at least 10 aminoacids containing the amino acid sequenceasparagine-proline-X-(phosphorylated)tyrosine, where X refers to anyamino acid. Numerous proteins having APB recognition domains are knownin the art, see for example Campbell et al., Proc. Natl. Acad. Sci. USA91:6344, 1994, hereby incorporated by reference herein. Kavanaugh andWilliams Science 266:1862, 1994 (not admitted to be prior art), identifya protein domain present in Shc that specifically binds to thetyrosine-phosphorylated form of its target at a domain other than SH2sequences.

The present invention provides a target site for designing therapeuticand diagnostic agents. The present disclosure allows for the design oftherapeutic agents able to modulate APB mediated activity betweenproteins and, thus alter signal transduction. Preferred modulatingagents can decrease signal transduction from a receptor tyrosine kinaseby disrupting binding involving an APB domain.

Thus, a first aspect of the present invention features a method fortreating a disease or disorder in an organism characterized by anabnormal level of interaction between an APB domain and its bindingpartner. The disease or disorder may also be characterized by anabnormality in a signal transduction pathway, wherein the pathwaycontains a protein with an APB domain. The method includes disrupting orpromoting that interaction (or signal) in vivo. The method also involvesinhibiting the activity of the complex formed between the APBdomain-containing protein and its binding partner.

By “organism” is meant any living creature. The term includes mammals,and specifically humans.

An “abnormal level” of interaction refers to a different level ofinteraction than occurring in the general population of healthyorganisms. The abnormal level can be an increased amount or a decreasedamount. The abnormality in signal transduction may be realized as anabnormality in cell growth, migration or other function.

By “signal transduction pathway” is meant the sequence of events thatinvolves the transmission of a message from an extracellular protein tothe cytoplasm through a cell membrane. The signal ultimately will causethe cell to perform a particular function, for example, to proliferateand therefore cause cancer. Various mechanisms for the signaltransduction pathway (Fry et al., 1993, Protein Science, 2:1785-1797)provide possible methods for measuring the amount or intensity of agiven signal.

Depending upon the particular disease associated with the abnormality ina signal transduction pathway, various symptoms may be detected. Forexample, if the disease is breast cancer, one may detect cellproliferation or tumor size, among other symptoms.

Furthermore, since some adaptor molecules recruit secondary signaltransducer proteins towards the membrane, one measure of signaltransduction is the concentration and localization of various proteinsand complexes. In addition, conformational changes involved in thetransmission of a signal may be observed using circular dichroism andfluorescence studies.

Another aspect of the present invention describes a method for treatinga patient having a disease or disorder characterized by APB bindinginvolving the step of administering to the patient a therapeuticallyeffective amount of an agent which decreases binding between an APBrecognition region present in a first protein and an APB domain presentin a second protein. A “patient” refers to human, who preferably has acell proliferative disorder involving a receptor tyrosine kinase.

Preferably, the first protein is a receptor tyrosine kinase, the secondprotein is Shc, and the agent decreases one or more activities of thereceptor tyrosine kinase by decreasing signal transduction from Shc.More preferably, the receptor tyrosine kinase is either EGF, HER-2, orTrkA.

The agent decreases APB mediated activity. The effect on APB mediatedactivity can be determined using standard techniques which depend on thetype of activity being mediated. For example, different activities ofEGF, HER-2, or TrkA can be measured downstream of Shc binding.

A “therapeutic effective amount” generally refers to an amount whichinhibits, to some extent, growth of cells causing or contributing to acell proliferative disorder and brings about a therapeutic effect in ahuman.

“Cell proliferative disorders” refer to disorders wherein unwanted cellproliferation of one or more subset(s) of cells in a multicellularorganism occurs, resulting in harm (e.g., discomfort or decreased lifeexpectancy) to the multicellular organism. Cell proliferative disorderscan occur in different types of animals and in humans. Cellproliferative disorders include cancers, blood vessel proliferativedisorders, and fibrotic disorders.

A therapeutic effect relieves to some extent one or more of the symptomsof a cell proliferative disease or disorder. In reference to thetreatment of a cancer, a therapeutic effect refers to one or more or thefollowing: 1) reduction in tumor size; 2) inhibition (i.e., slowing tosome extent, preferably stopping) of tumor metastasis; 3) inhibition, tosome extent, of tumor growth; and 4) relieving to some extent one ormore of the symptoms associated with the disease or disorder.

In reference to treating a cell proliferative disease or disorder otherthan a cancer, a therapeutic effect refers to one or more of thefollowing: 1) the inhibition, to some extent, of the growth of cellscausing the disease or disorder; 2) the inhibition, to some extent, ofthe production of factors (e.g., growth factors) causing the disorder;and 3) relieving to some extent one more or the symptoms associated withthe disease or disorder. The disease or disorder may be characterized byan abnormality in the signal transduction pathway even if the level ofinteraction between the APB domain and its binding partner is normal.

“Characterized by APB binding” refers to the involvement of APB bindingin causing or accentuating to some extent, the disease or disorder. Forexample, APB binding of Shc can lead to phosphorylation of Shc which inturn can interact with Ras leading to transformation of a cell therebycausing cancerous growth. Shc activation may also aid in thetransformation of cells by other mechanisms and may exert other harmfuleffects.

Agents able to modulate APB mediated activity are generally targeted tomodulate APB mediated activity in one or more of the following ways: (1)by binding to the APB domain thereby inhibiting subsequent proteinbinding; (2) by binding to an APB recognition region thereby inhibitingsubsequent protein binding; (3) by binding to the APB domain andproducing either an increase or decrease in signal transduction; and (4)by binding to an APB recognition region and producing either an increaseor decrease in signal transduction. Examples of such agents includeorganic molecules, preferably 150 to 1,000 daltons, and polypeptides orantibodies able to bind to the APB domain or APB recognition region.

Another aspect of the present invention features a method for screeningfor an agent useful for treatment of disease or disorder characterizedby abnormal APB binding. The method involves assaying potential agentsfor the ability to disrupt or promote that interaction. The screeningmay also involve assaying potential agents for the ability to remove orreduce an abnormality in a signal transduction pathway, wherein thesignal transduction pathway contains a protein with an APB domain.

Another aspect of the present invention describes a method fordiagnosing a disease or disorder characterized by an abnormal level ofAPB mediated activity. The method includes the step of detecting thelevel of APB binding between an APB recognition region present in afirst protein and an APB domain present in a second protein.

Assays to detect the level of APB binding can be carried out usingdifferent techniques. For example, cells can be isolated from a patientand the level of interaction can be measured using a competitive ornon-competitive assay formats involving an APB recognition binding agentor an APB domain binding agent.

Another aspect of the present invention describes a method of assayingfor agents useful for disrupting APB interactions or in the diagnosis ofAPB diseases or disorders. The method involves the step of measuring theability of an agent to bind to an APB recognition region or an APBdomain.

Another aspect of the present invention describes a purified recombinantpolypeptide encoding a APB domain having at least 20% sequence identityor 30% sequence similarity to the APB domain present in Shc. By“recombinant” is meant that the APB domain is present on a polypeptidefragment. The polypeptide fragment is obtained from a particular sourcesuch as Shc or synthetically produced, contains an APB region having atleast 20% sequence identity or 30% sequence similarity to the Shc APBregion, and is less than the full length Shc. Thus, the presentinvention also features a peptide comprising, consisting or consistingessentially of an APB domain.

By “purified” is meant that the polypeptide is in a form (i.e., itsassociation with other molecules) distinct from naturally occurringpolypeptide. Preferably, the polypeptide is provided as a substantiallypurified preparation representing at least 75%, more preferably at least85%, most preferably at least 95% of the total protein in thepreparation.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cloning of murine p₅₂ ^(Shc). A, binding of EGF receptor probeto Grb2 or Grb12 expressing λEXlox phages. Arrows indicate examples ofpositive plaques. B, amino acid sequence alignment of human and murinep52^(Shc). Dots indicate identical residues; dashed line indicates a gapin the murine sequence.

FIGS. 2A and 2B. Association of GST-Shc 1-209 with the EGF Receptor(EGFR). A, HER14 lysates from either EGF-stimulated (+) or unstimulated(−) cells (5×10⁶ cells) were incubated with 3 μg of GST fusion proteinimmobilized on glutathione-agarose beads. The bound proteins werewashed, subjected to SDS-PAGE, transferred to nitrocellulose andimmunoblotted with anti-EGF receptor antibody, anti-C (anti-EGFR). HER14lysate (4×10⁵ cells) was run directly on the gel in lanes 5 and 6. B,unstimulated HER14 lysates (5×10⁶ cells) were immunoprecipitated withanti-EGFR antibody (mAb 108). The immobilized receptor was thenphosphorylated (+) by the addition of 5 mM MnCl₂ and 50 μM ATP or leftunphosphorylated (−) by adding MnCl₂ alone. Bacterial lysates containing5 μg of GST, GST-Grb7, and GST-Shc 1-209 were then diluted in 1 ml of 1%Triton X-100 lysis buffer, added to the immobilized receptor andincubated at 4° C. for 90 min. The bound proteins were washed andtogether with 40 μl of the post-binding supernatant separated bySDS-PAGE and transferred to nitrocellulose. The bound fractions (leftpanel) and supernatants (right panel) were immunoblotted with anti-GST.

FIG. 3. Association of GST-Shc 1-209 with mutant forms of the EGFreceptor. NIH 3T3 cells overexpressing various EGF receptor mutants werestimulated with EGF (200 ng/ml) for 3 min and then lysed. Wild type EGFreceptor (W.T.) contains all five known EGF receptor autophosphorylationsites, Y2F has Tyr-992 and Tyr-1068 mutated to phenylalanine, Y5F hasall five known EGF receptor autophosphorylation sites mutated tophenylalanine, and CD196 has a carboxyterminal deletion of 196 aminoacids deleting all known autophosphorylation sites (Li, N., et al.(1994) Oncogene (In Press). Beads containing GST-Shc 1-209 fusionprotein (3 μg) were added to the various lysates containing equalamounts of the receptor (left panels) or the lysates wereimmunoprecipitated with the anti-EGF receptor (EGFR) antibody (mAb108)(right panels). All samples were incubated at 4° C. for 90 min. Thebound proteins were then washed, subjected to SDS-PAGE and transferredto nitrocellulose. The membranes were immunoblotted withanti-phosphotyrosine (anti-Ptyr) antibody (upper panels) and anti-EGFR(anti-F; lower panels). IP, immunoprecipitate.

FIGS. 4A, 4B, 4C and 4D. Binding of Shc fragments to growth factorreceptors. A, schematic diagram illustrating the Shc fragments used inbinding studies. Full length murine p52^(shc) is indicated in (i) while(ii)-(vi) were generated as GST fusion proteins for use in receptorbinding studies. B, association of various GST fusion proteins withactivated EGF receptor (EGFR). Binding was performed as in FIG. 2A withthe exception that all lysates were stimulated with EGF. All studiesused 3 μg of fusion proteins except for the Shc SH2 where 10 μg wasused. Immunoblotting was performed with anti-phosphotyrosine. C, bindingof Shc fusion proteins to NIH 3T3 cells expressing a HER1/2 chimera.This chimera consists of the EGF receptor extracellular binding domainand the intracellular region of HER2/neu. After stimulation of thesecells with EGF (200 ng/ml) lysates were prepared and binding as well asblotting studies were performed as in B using anti-phosphotyrosineantibody. D, binding of Shc 1-209 to TrkA. Beads containing 10 μg of GSTfusion proteins Shc 1-209, phospholipase C-γ (PLC-γ) SH2 (contains bothN and C SH2 domains), or GST alone were added to lysates from 2×10⁷ PC12cells stimulated with 50 ng/ml nerve growth factor (lanes 1, 2, and 4).The binding of the same amount of lysate to 2 μg of anti-TrkA antibodyis shown as a positive control and to indicate the position of TrkA onthe blot (lane 3). Otherwise, the binding and blotting were performed asin B using anti-phosphotyrosine antibody.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention identifies the APB domain as a target site fordiagnostic agents, and therapeutic agents able to modulate one or moreAPB mediated activities. Different diseases or disorders involving APBdomain binding between proteins can be targeted by the presentinvention. The therapeutic agents can modulate signal transduction and,thereby, can be used to treat diseases or disorders where signaltransduction plays a role. For example, the interaction of the APBdomain of Shc with EGF, TrkA, and HER-2 is described below. Bydisrupting such interaction diseases associated with EGF, TrkA, or HER-2activation can be treated.

I. Targeted Diseases

The identification of the APB domain and its ability to play a role insignal transduction allows for targeting a wide range of diseases ordisorders associated with tyrosine kinase activity. Examples of proteinsbinding the APB domain include EGF, HER-2 and TrkA. These proteins arereceptor tyrosine kinases which have different diseases or disordersassociated with their activation and subsequent signal transduction. Bymodulating APB domain binding to such receptor tyrosine kinases theassociated disease can be treated. Examples of diseases associated withEGF, HER-2 and TrkA are described in sections IA-IC infra.

Additionally, a disorder involving a protein-protein complex may, forexample, develop because the presence of such a complex brings about theaberrant inhibition of a normal signal transduction event. In such acase, the disruption of the complex would allow the restoration of theusual signal transduction event.

Further, an aberrant complex may bring about an altered subcellularadaptor protein localization, which may result in, for example,dysfunctional cellular events. An inhibition of the complex in this casewould allow for restoration or maintenance of a normal cellulararchitecture. Still further, an agent or agents that cause(s) disruptionof the complex may bring about the disruption of the interactions amongother potential components of a complex.

Additional diseases or disorders which can be targeted by the presentinvention can be identified using the present disclosure as guide. BothAPB domain containing proteins and APB recognition region proteins canbe identified. For example, the identification of Shc as containing anAPB domain is described in the Example below. Additional proteinscontaining an APB domain can be obtained by repeating the procedure orby equivalent methods. Proteins containing an APB recognition domain canbe identified using a similar procedure starting with a labeled APBdomain binding protein. For example, a labeled Shc protein.

A. EGFR Cell Proliferation Disorders

EGFR cell proliferation disorders are characterized by inappropriateEGFR activity. “Inappropriate EGFR” activity refers to either 1)EGF-receptor (EGFR) expression in cells which normally do not expressEGFR; 2) EGF expression by cells which normally do not express EGF; 3)increased EGF-receptor (EGFR) expression leading to unwanted cellproliferation; 4) increased EGF expression leading to unwanted cellproliferation; and/or 5) mutations leading to constitutive activation ofEGF-receptor (EGFR). The existence of inappropriate or abnormal EGF andEGFR levels or activities can be determined by procedures well known inthe art.

An increase in EGF activity or expression is characterized by anincrease in one or more of the activities which can occur upon EGFligand binding such as: (1) auto-phosphorylation of EGFR, (2)phosphorylation of an EGFR substrate (e.g., PLCγ, see Fry supra), (3)activation of an adapter molecule, and/or (4) increased cell division.These activities can be measured using techniques known in the art. Forexample auto-phosphorylation of EGFR can be measured using ananti-phosphotyrosine antibody, and increased cell division can beperformed by measuring ³H-thymidine incorporation into DNA. Unwantedcell proliferation can result from inappropriate EGFR activity occurringin different types of cells including cancer cells, cells surrounding acancer cell, and endothelial cells. Examples of disorders characterizedby inappropriate EGF activity include cancers such as glioma, head,neck, gastric, lung, breast, ovarian, colon, and prostate; and othertypes of cell proliferative disorders such as psoriasis.

B. HER2 Cell Proliferation Disorders

HER2 cell proliferation disorders are characterized by over-activity ofHER2. Over-activity of HER2 refers to either an amplification of thegene encoding HER2 or the production of a level of HER2 activitycorrelated with a cell proliferative disorder (i.e., as the level ofHER2 increases the severity of one or more of the symptoms of the cellproliferative disorder increases).

Activation of HER-2 protein can be caused by different events such asligand-stimulated homo-dimerization, ligand-stimulatedhetero-dimerization and ligand-independent homo-dimerization.Ligand-stimulated hetero-dimerization appears to be induced by EGFR toform EGF-R/HER-2 complexes and by neu differentiation factor/heregulin(NDF/HRG) to form HER-2/HER-3 and/or HER-2/HER-4 complexes. Wada et al.,Cell 61:1339, 1990; Slikowski et al., J. Biol. Chem. 269:14661, 1994;Plowman et al., Nature 266:473, 1993. Ligand-dependent activation ofHER2 protein is thought to be mediated by neu-activating factor (NAF)which can directly bind to p185(HER-2) and stimulate enzymatic activity.Dougall et al., Oncogene 9:2109, 1994; Samata et al., Proc. Natl. Acad.Sci. USA 91:1711, 1994. Ligand-independent homo-dimerization of HER2protein and resulting receptor activation is facilitated byover-expression of HER2 protein.

HER2 activity can be assayed by measuring one or more of the followingactivities: (1) phosphorylation of HER2; (2) phosphorylation of a HER2substrate; (3) activation of an HER2 adapter molecule; and (4) increasedcell division. These activities can be measured using techniquesdescribed known in the art.

Patients suffering from a HER2 driven disease or disorder can beidentified by analysis of their symptoms by procedures well known tomedical doctors. Such identified patients can then be treated asdescribed herein.

HER2 driven diseases or disorders are typically cell proliferativedisorders such as cancers. HER2 driven disorders appear to beresponsible for a sub-population of different types of cancers. Forexample, Slamon et al., Science 244:707, 1989, examined the correlationbetween HER-2/neu and breast and ovarian carcinoma, and also examinedprocedures used to measure the correlation. According to Slamon:

The HER-2/neu proto-oncogene is amplified in 25 to 30 percent of humanprimary breast cancers and this alteration is associated with diseasebehavior. In this report, several similarities were found in the biologyof HER-2/neu in breast and ovarian cancer, including a similar incidenceof amplification, a direct correlation between amplification andover-expression, evidence of tumors in which overexpression occurswithout amplification, and the association between gene alteration andclinical outcome. A comprehensive study of the gene and its products(RNA and protein) was simultaneously preformed on a large number of bothtumor types. This analysis identified several potential shortcomings ofthe various methods used to evaluate HER-2/neu in these diseases(Southern, Northern, and Western blots, and immunohistochemistry) andprovided information regarding considerations that should be addressedwhen studying a gene or gene product in human tissue. The data presentedfurther support the concept that the HER-2/neu gene may be involved inthe pathogenesis of some human cancers.

The use of the present, invention to treat breast cancer is preferredbecause of the prevalence and severity of breast cancer. Carcinoma ofthe breast is the most common cancer among women and their secondleading cause of cancer death (Marshall, E., Science 259:618-621, 1993).The incidence of breast cancer has been increasing over the past severaldecades (Marshall, supra, and Harris, J. R., et al, New Engl. J. Med.,327(5):319-328, 1992). In addition to breast cancers, increased HER2activity or gene expression has been associated with certain types ofstomach adenocarcinomas, salivary gland adenocarcinomas, endometrialcancers, ovarian adenocarcinomas, gastric cancers, colorectal cancers,non-small cell lung cancer, and glioblastomas.

C. Trk Cell Proliferation Disorders

Differentiation and survival of neuronal cells is mediated, in part, bythe activity of a family of related receptor tyrosine kinases, includingTrkA, TrkB, and TrkC and ligands such as nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF) and neuro-trophins-3 and 4(NT-3 and NT-4). Barbacid, M., Biochimica et Biophysica Acta1072:115-127, 1991, Kaplan, D. R., Science 252:554-558, 1991; Klein, R.,Cell 65:189-197, 1991; Davies, A. M., Nature 368:193-194, 1994 andKlein, R., Cell 66:395-403, 1991. Examples of diseases or disorders tobe treated or diagnosed by targeting Trk include Alzheimer's disease,Parkinson's disease, Lou Gerhig's disease (ALS), trauma, damaged orsevered nerve injuries, Huntington's chorea, multiple schleroris,muscular dystrophy, syringomiplia, Tabes Dorsalis, and cardiovascularaccidents. These and other diseases or disorders are often characterizedby one or more of the following symptoms: atasia, aphasia, paralysis,paresea, and paralgies.

Abnormalities in Trk signal transduction pathways can lead to variousdiseases through both underactivity and over-activity. Examples ofdisorders which are characterized by underactivity of a signaltransduction pathway include various neurodegenerative diseases such asmyasthenia gravis, amyotrophic lateral sclerosis, cervical spondylosis,and Alzheimer's disease. A neurological disease possibly characterizedby over-activity of a signal transduction pathway is neurofibromatosis.See, in general, The Merck Manual of Diagnosis and Therapy, 16thEdition, 1992.

Mutations in the Trk gene are also involved in cancer. Mutant Trk geneshave been isolated from both colon and thyroid carcinomas. In general,the mutations involve a molecular rearrangement such that thetrans-membrane and cytoplasmic portions of the Trk gene remain intact,however, the normal extracellular ligand binding sequences have beenreplaced by foreign sequences such as those coding for tropomyosin.Martin-Zanca, D., Nature 319:743-748, 1986, Pulciani, S., Nature300:539-542, 1982, Bongarzone, I., Oncogene 4:1457-1462, 1989 and Park,M., Cell 45:895-904, 1986. Such mutations may lead to abnormal signaltransduction activity by stimulating activity even in the absence of thenormal ligand and/or stimulating activity in an inappropriate(non-neuronal) cell type.

The importance of the Trk gene family for the growth, differentiationand survival of nerve cells was recently demonstrated by experiments inmice. Gene targeting was used to generate mice with null mutations ingenes encoding each of the known Trk receptors, and in one case the geneencoding a Trk ligand. Smeyne, R. J., et al., Nature 368:246-249, 1994,and Klein, R., et al., Nature 268:249-251, 1994. Such mice cannotexpress the target gene and all had severe neurological dysfunction andimportant types of neural tissue were absent.

The development and maintenance of cellular communication networkswithin the central and peripheral nervous system is regulated byneurotrophic factors, which through activation of specific cell surfacereceptors generate differentiation and survival signals in neuronal celltypes. Binding of the neurotrophic factor to its receptor initiates acellular signal transduction cascade involving diverse cytoplasmiccomponents eventually resulting in a specific nuclear response. Specificcellular responses in nerve cells can include, for example, neuriteoutgrowth, acquisition of Na⁺-based action potential and cell survivalin serum-free medium.

The complex processes of cell growth, differentiation and cell survivalare mediated by diverse and divergent signal transduction pathways. Themultiple phosphorylated tyrosines found in activated receptor tyrosinekinases serve as binding sites for different signaling components, whichin turn modulate the transduction of a signal along a particularpathway. In the case of the NGF receptor Trk, specific phosphorylatedtyrosines within the cytoplasmic portion of the receptor can bind to thesignaling components phospholipase C-gamma (PLC-gamma), Shc and thenon-catalytic subunit of phosphatidylinositol-3′-kinase, p85. Obermeier,A., EMBO Journal 12:933-941, 1993 and Obermeier, A., J. Biol. Chem.268:22963-22966, 1993.

These proteins in turn each stimulate different and distinct furtherdownstream signaling components until instructions are finallytransmitted to the cell nucleus. For example, Shc binds to the Grb2/SOScomplex, which in turn allows the activation of ras.

II. Therapeutic Agents

Different types of therapeutics agents can be used to modulate APBmediated activity such as organic molecules, inorganic molecules, andpolypeptides binding to the APB domain or APB region site. Such agentscan be obtained and administered using the present disclosure as aguide. For example, APB binding molecules such as organic or inorganicmolecules, preferably organic molecules can be obtained as described insection III infra.

A. Polypeptides Modulating APB Mediated Activity

Therapeutic protein or polypeptide agents can be designed to bind to anAPB domain or an APB recognition region. Polypeptides binding to an APBrecognition region preferably are at least 10 amino acid in length andcontain the amino acid sequenceasparagine-proline-X-(phosphorylated)tyrosine, where X refers to anyamino acid.

The terms “protein” and “polypeptide” refers to 5 or more amino acidsjoined together by peptide bonds. An “amino acid” is a subunit that ispolymerized to form proteins and there are twenty amino acids that areuniversally found in proteins. The general formula for an amino acid isH₂N—CHR—COOH, in which the R group can be varies from a hydrogen atom(as in the amino acid glycine) to a complex ring (as in the amino acidtryptophan).

APB amino acid recognition regions binding to an APB recognition regionare preferably at least 150 amino acids in length and contain an aminoacid sequence having at least 20% sequence identity or at least 30%sequence similarity to the APB domain present in Shc. In a preferredembodiment an APB domain has at least 40%, at least 65%, or at least 80%sequence identity to the APB domain present in Shc. In another preferredembodiment the APB domain has at least 50%, at least 75%, or at least90% sequence similarity to the APB domain present in shc.

The percentage of sequence identity between two domains is calculated bydividing the number of amino acids that are the same in a given regionby the total number of amino acids in the given region. Proteins havingsuch domains are readily identified using standard protocols. Thepercentage of sequence similarity can calculated using a computerprogram (such as the GCG Bestfit program) that scores a protein basedupon the number of gaps that must be induced to achieve similarity.Regions that have at least 20% identity or at least 30% similarity arerecognized as containing the same domain independent of any knowledge ofthe function of the proteins or domains. However, when knowledgeregarding the function of the proteins or domain is known, thenequivalent domains may be identified with much lower identity orsimilarity. For example, the pleckstrin domain contains two proteinsthat only share approximately 5% identity to each other. Musacchio,supra.

B. Molecules Modulating APB Mediated Activity

Inorganic and organic molecules able to modulate APB mediated activitymay also be used to treat diseases or disorders involving APB mediatedactivity. Such molecules can be obtained as described in section IIIinfra.

C. Antibodies Able to Modulate APB Mediated Activity

Antibodies capable of binding to an APB domain or APB receptorrecognition region can be used for treating diseases or disordersinvolving APB binding. For example, nucleotide sequences encodingsingle-chain antibodies targeted to an APB domain or APB receptorrecognition region may be expressed within the target cell population byutilizing, for example, techniques such as those described in Marasco etal. Proc. Natl. Acad. Sci. USA 90:7889-7893, 1993.

Antibodies which can be used as therapeutic or diagnostic agents includepolyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, fragments produced by a FAb expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Antibodies may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. Suchantibodies may be used, for example, in the detection of aprotein-protein complex in a biological sample, or, alternatively, as amethod for the inhibition of a complex formation, thus, inhibiting thedevelopment of a disease or disorder.

Protein-protein complexes are formed between at least a proteincontaining an APB domain and a protein containing an APB recognitionregion. Under standard physiological conditions, the components of suchcomplexes are capable of forming stable, non-covalent attachments withone or more of the other complex components.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a protein-protein complex, or individual components. For theproduction of polyclonal antibodies, various host animals may beimmunized by injection with the antigen such as rabbits, mice, and rats.Various adjuvants may be used to increase the immunological response,depending on the host species, such as Freund's (complete andincomplete), mineral gels (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol), and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

A monoclonal antibody, which is a substantially homogeneous populationof antibodies to a particular antigen, may be obtained by techniquesproviding for the production of antibody molecules by continuous celllines in culture. Examples of such techniques include the hybridomatechnique described by Kohler and Milstein, Nature 256:495-497 (1975)and U.S. Pat. No. 4,376,110, the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983 Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

The hybridoma producing the mAb may be cultivated in vitro or in vivo.Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to producecomplex-specific single chain antibodies. Single chain antibodies areformed by linking the heavy and light chain fragment of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments containing specific binding sites of a complex may begenerated by known techniques. For example, such fragments include theF(ab′)₂ fragments which can be produced by pepsin digestion of theantibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries may be constructed (Huse et al., 1989, Science,246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity to the receptor tyrosinekinase/adaptor complex.

III. Identification of APB Modulating or Binding Agents

APB binding complexes, proteins containing APB domains and proteinscontaining APB recognition regions can be used to obtain APB modulatingor binding agents. Methods for purifying and/or producing suchcomplexes, components of the complexes (i.e., containing a proteinhaving an APB domain and a protein having an APB recognition region) aredescribed herein. Antibodies to such APB binding complexes, proteinscontaining APB domains and proteins containing APB recognition regionscan be obtain as described in section IIC, supra.

APB binding agent may include peptides made of D- and/or L-configurationamino acids (in, for example, the form of random peptide libraries; seeLam, K. S. et al., 1991, Nature 354:82-84), phosphopeptides (in, forexample, the form of random or partially degenerate, directedphosphopeptide libraries, see Songyang, Z. et al., 1993, Cell 767-778),antibodies, and small organic or inorganic molecules. Syntheticcompounds, natural products, and other sources of potentiallybiologically active materials may be screened in a variety of ways, asdescribed herein. The compounds, antibodies, or other moleculesidentified may be used as oncogenic disorder treatments.

One method for identifying an agent able to modulate APB mediatedactivity and/or bind to an APB domain or recognition region involves thefollowing steps:

(a) exposing at least one agent to a protein comprising an APB domain oran APB recognition region for a time sufficient to allow binding of theagent;

(b) removing non-bound agents; and

(c) determining the presence of bound agent.

By utilizing this procedure, large numbers of types of molecules may besimultaneously screened for complex component-binding activity.

This approach can be pursued using different techniques. Such asattaching the protein containing the APB domain or recognition region toa solid matrix, such as agarose or plastic beads, microtiter wells,petri dishes, or membranes composed of, for example, nylon ornitrocellulose. The protein can be attached to solid matrix usingstandard techniques such as by using a component specific antibody bounddirectly to the solid support.

Molecules exhibiting binding activity may be further screened for anability to effect APB binding or modulate APB mediated activity. Forexample, the molecule can be tested for its ability to increase ordecrease APB binding. Alternatively, the molecule may be tested for itsability to increase or decrease one or more activities mediated by theAPB domain protein.

In another assay, molecules may be directly screened for an ability topromote the APB binding complexes. For example, in vitro complexformation may be assayed by, first, immobilizing a polypeptidecontaining an APB domain or APB recognition region to a solid support.Second, the immobilized polypeptide is exposed to a potential binding ormodulating agent. Third, the ability of the second component (i.e., apolypeptide containing an APB domain or APB recognition region not boundto the support) to form a complex with the immobilized component in thepresence of the agent is measured. In addition, one could look for anincrease in binding.

Additionally, complex formation in a whole cell may be assayed byutilizing co-immunoprecipitation techniques well known to those ofordinary skill in the art. Briefly, a cell line capable of forming acomplex is exposed to a potential modulating or binding agent, and acell lysate is prepared from this exposed cell line. An antibody raisedagainst one of the components of the complex is added to the celllysate, and subjected to standard immunoprecipitation techniques. Incases where a complex is still formed the immunoprecipitation willprecipitate the complex, whereas in cases where the complex has beendisrupted only the complex component to which the antibody is raisedwill be precipitated.

A preferred method for assessing modulation of complex formation withina cell utilizes a method similar to that described above. Briefly, acell line capable of forming am APB binding complex is exposed to a testagent. The cells are lysed and the lysate contacted with an antibodyspecific to one component of the complex, the antibody having beenpreviously bound to a solid support. Unbound material is washed away,and the bound material is exposed to a second antibody bindingspecifically to a second component of the complex. The amount of secondantibody bound is easily detected by techniques well known in the art.Cells exposed to an inhibitory test agent will have formed a lesseramount of complex compared to cells not exposed to the test agent, asmeasured by the amount of second antibody bound. Cells exposed to a testagent that promotes complex formation will have an increased amount ofsecond antibody bound.

The effect of an agent on the transformation capability of an APBbinding complex may be directly assayed. Such agents include thoseagents identified by utilizing the above screening technique. Forexample, an agent may be administered to a cell such as a breast cancercell. The transformation state of the cell may then be measured invitro, by monitoring, for example, its ability to form colonies in softagar. Alternatively, a cell's transformation state may be monitored invivo by, for example, determining its ability to form tumors inimmunodeficient nude or severe combined immunodeficiency (SCID) mice.

IV. Purification and Production of Polypeptides

This section describes methods for the synthesis or recombinantexpression of polypeptides containing APB domains or APB recognitionregion. Also described are methods for which cells exhibiting theprotein may be expressed.

A. Synthesis and Expression Methods

Methods for the synthesis of polypeptides are well-known to those ofordinary skill in the art. See, for example, Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman and Co., NY, which isincorporated herein, by reference, in its entirety.

Components of a complex which have been separately synthesized orrecombinantly produced, may be reconstituted to form a complex bystandard biochemical techniques. For example, samples containing thecomponents of the complex may be combined in a solution buffered withgreater than about 150 mM NaCl, at a physiological pH in the range of 7,at room temperature. For example, a buffer comprising 20 mM Tris-HCl, pH7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5%deoxycholate and 2 mM EDTA could be used.

Polypeptides containing an APB domain or APB recognition region may alsobe produced using expression vectors encoding the polypeptide. Theexpression vectors should contain protein coding sequences andappropriate transcriptional/translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. DNA and RNAsynthesis may, additionally, be performed using an automatedsynthesizers. See, for example, the techniques described in Maniatis etal., 1989, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. and Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y.

A variety of host-expression vector systems may be utilized to expressencoded polypeptides. Such host-expression systems represent vehiclesand cells by which the encoded sequence of interest may be produced.Examples include microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing protein coding sequences; yeast(e.g., Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the protein coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the protein coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing theprotein coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the clonedprotein. For example, when large quantities of proteins are to beproduced for the generation of antibodies or to screen peptidelibraries, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J. 2:1791), in which the protein coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 264:5503-5509).

pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned proteincan be released from the GST moiety.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the complex coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingproteins in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Specific initiation signals may also berequired for efficient translation of inserted coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, and transcription terminators. (seeBittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include CHO, VERO, BHK,HeLa, COS, MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably coexpressproteins may be engineered. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withthe protein encoding DNA independently or coordinately controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, and polyadenylation sites), and aselectable marker.

Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which coexpress both theAPB domain protein and APB recognition protein. Such engineered celllines are particularly useful in screening and evaluation of agents thataffect signals mediated by the complexes.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981), Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

B. Derivatives of Shc APB domain

Also provided herein are functional derivatives of an APB domain presentin full length Shc. By “functional derivative” is meant a “chemicalderivative,” “fragment,” “variant,” “chimera,” or “hybrid” of the APBdomain protein. A functional derivative retains at least a portion ofthe function of the protein, for example reactivity with an antibodyspecific for the protein, enzymatic activity or binding activitymediated through noncatalytic domains.

A “chemical derivative” contains additional chemical moieties notnormally a part of the protein. Covalent modifications of the proteinmay be introduced by reacting targeted amino acid residues of thepeptide with an organic derivatizing agent capable of reacting withselected side chains or terminal residues.

For example, cystienyl residues most commonly are reacted withalpha-haloacetates (and corresponding amines), such as chloroacetic acidor chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineε-amino group.

Tyrosyl residues are well-known targets of modification for introductionof spectral labels by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction carbodiimide (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residue are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful, for example, forcross-linking the component peptides of the complexes to each other orthe complex to a water-insoluble support matrix or to othermacromolecular carriers. Commonly used cross-linking agents include, forexample, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Such derivatized moieties may improve the stability, solubility,absorption, and/or biological half life. The moieties may alternativelyeliminate or attenuate an undesirable side effect of a protein agent.Moieties capable of mediating such effects are disclosed, for example,in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,Easton, Pa. (1990).

The term “fragment” is used to indicate a polypeptide derived from thefull length amino acid sequence of a protein. Such a fragment may, forexample, be produced by proteolytic cleavage of the full-length proteinor using recombinant techniques. Preferably, the fragment is obtainedrecombinantly by appropriately modifying the DNA sequence encoding theproteins to delete one or more amino acids at one or more sites of theC-terminus, N-terminus, and/or within the native sequence. Fragments ofa protein, when present in a complex resembling the naturally occurringcomplex, are useful for screening for agents that act to modulate signaltransduction. It is understood that such fragments, when present in acomplex may retain one or more characterizing portions of the nativecomplex. Examples of such retained characteristics include: substratespecificity, interaction with other molecules in the intact cell,regulatory functions, or binding with an antibody specific for thenative complex, or an epitope thereof.

Another functional derivative within the scope of the present inventionis a complex comprising at least one “variant” polypeptide which eitherlack one or more amino acids or contain additional or substituted aminoacids relative to the native polypeptide. The variant may be derivedfrom a naturally occurring complex component by appropriately modifyingthe protein DNA coding sequence to add, remove, and/or to modify codonsfor one or more amino acids at one or more sites of the C-terminus,N-terminus, and/or within the native sequence. It is understood thatsuch variants having added, substituted and/or additional amino acidsretain one or more characterizing portions of the native complex, asdescribed above.

A functional derivative of complexes comprising proteins with deleted,inserted and/or substituted amino acid residues may be prepared usingstandard techniques well-known to those of ordinary skill in the art.For example, the modified components of the functional derivatives maybe produced using site-directed mutagenesis techniques (as exemplifiedby Adelman et al., 1983, DNA 2:183) wherein nucleotides in the DNAcoding the sequence are modified such that a modified coding sequence ismodified, and thereafter expressing this recombinant DNA in aprokaryotic or eukaryotic host cell, using techniques such as thosedescribed above. Alternatively, components of functional derivatives ofcomplexes with amino acid deletions, insertions and/or substitutions maybe conveniently prepared by direct chemical synthesis, using methodswell-known in the art. The functional derivatives of the complexestypically exhibit the same qualitative biological activity as the nativecomplexes.

V. Diagnosis

The present invention also describes assays which can be used todiagnosis diseases or disorders involving APB mediated activity.Diagnosis can be carried out, for example, by measuring the level ofinteraction between a protein having an APB domain and a protein havingan APB recognition region; measuring the amount of APB domain proteinpresent; and/or measuring the amount of APB recognition region proteinpresent. The measured amounts of these different indicators can becompared to that occurring in healthy individuals and in individualssuffering from a cell proliferative disease or disorder to determine theindicator level associated with the disease or disorder.

Protein complexes involving APB binding may be utilized in a prognosticevaluation of the condition of a patient suspected of exhibiting such adisorder. For example, biological samples obtained from patientssuspected of exhibiting a disorder involving a protein complex may beassayed for the presence and amount of such complexes. If such a proteincomplex is normally present, and the development of the disorder iscaused by an abnormal quantity of the complex, the assay should comparecomplex levels in the biological sample to the range expected in normaltissue of the same cell type.

Alternatively, one or more of the components of the protein complex maybe present in an abnormal level or in a modified form, relative to thelevel or form expected in normal, nononcogenic tissue of the same celltype. It is possible that overexpression of both components of an APBcomplex may indicate a particularly aggressive disorder.

Thus, an assessment of the individual levels of APB domain protein, APBrecognition protein, and mRNA encoding such proteins, in diseased tissuecells may provide valuable clues as to the course of action to beundertaken in treatment. Assays to measure nucleic acid encoding aprotein are well known to those of ordinary skill in the art, and mayinclude Northern blot analysis, RNAse protection assays, and PCR fordetermining mRNA levels. Assays determining protein levels are also wellknown to those of ordinary skill in the art, and may include Westernblot analysis, immunoprecipitation, and ELISA analysis. Each of thesetechniques may also reveal potential differences in the form (e.g., theprimary, secondary, or tertiary amino acid sequence, and/orpost-translational modifications of the sequence) of the component(s).

VI. Administration

Agents modulating APB activity can be administered to a patient usingstandard techniques. A particular agent can be administered by itself orin pharmaceutical compositions where it is mixed with suitable carriersor excipient(s). For example, small hydrophobic organic molecules may bedirectly administered intracellularly.

Toxicity and therapeutic efficacy of APB activity modulating agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Agents exhibiting large therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies canbe used to formulate a range of dosage for use in human. The dosage ofsuch agents lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays and animal models. For example, a dose can be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of the test agent which achieves a half-maximal modulationof APB mediated activity, measured for example by disruption of theprotein complex, or a half-maximal inhibition of the cellular leveland/or activity of a complex component). Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”,Ch. 1 p1). A physician of ordinary skill in the art would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, such a physician wouldalso know to adjust treatment to higher levels if the clinical responsewere not adequate (precluding toxicity).

The magnitude of an administrated dose in the management of theoncogenic disorder of interest will vary with the severity of thecondition to be treated. The severity of the condition may, for example,be evaluated, in part, by standard prognostic evaluation methods.Further, the dose and perhaps dose frequency, will also vary accordingto the age, body weight, and response of the individual patient. Aprogram comparable to that discussed above may be used in veterinarymedicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” 1990, 18th ed., Mack Publishing Co., Easton,Pa. Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, and intraocular injections. For injection,the agents of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

With proper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The agents can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the agents to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. Molecules present in an aqueous solution at thetime of liposome formation may be incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, aredelivered into the cell cytoplasm.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those of ordinaryskill in the art, especially in light of the detailed disclosureprovided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activeagents into preparations which can be used pharmaceutically.Pharmaceutical compositions may be manufactured using techniques such asconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active agents in water-soluble form.Additionally, suspensions of the active agents may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, and synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances increasing the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents increasing the solubility of the agents to allowfor the preparation of highly concentrated solutions.

The preparations formulated for oral administration may, for example bein the form of tablets, dragees, capsules, or solutions. Pharmaceuticalpreparations for oral use can be obtained by combining the active agentswith solid excipient, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipients are,in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations, for example, maizestarch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose; and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active agent doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active agents may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

VII. Gene Therapy

Gene therapy can be achieved by transferring a gene encoding an APBmodulating polypeptide agent into a patient in a manner allowingexpression of the polypeptide. Recombinant nucleic acid moleculesencoding polypeptide agents can be introduced into a cell in vivo or exvivo. In vivo transfection techniques include the use of liposomes andretroviral vectors. Miller, Nature 357: 455-460, hereby incorporated byreference herein. Ex vivo transfection increases the number of availabletransfection techniques, but also adds additional complications due toremoval and subsequent insertion of cells into a patient.

Nucleotide sequences encoding polypeptide agents which are to beutilized intracellularly may be expressed in the cells of interest,using techniques which are well known to those of ordinary skill in theart. For example, expression vectors derived from viruses such asretroviruses, vaccinia virus, adenoviruses, adeno-associated virus,herpes viruses, or bovine papilloma virus, may be used for delivery andexpression of such nucleotide sequences into the targeted cellpopulation. Methods for the construction of such vectors are well known.See, for example, the techniques described in Maniatis et al., 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y.; Ausubel et al., 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.; and Gluzaman,eds. Eukaryotic Viral, Cold Spring Harbor Laboratory, 1982.

VII. EXAMPLES

An example is described below illustrating different aspects andembodiments of the present invention. The text of the example is toappear as a Communication by Blaikie et al., Journal of BiologicalChemistry 269:32031-32034, 1994. The example is not intended in any wayto limit the disclosed invention.

Shc is a ubiquitously expressed Src homology 2 (SH2) (Src homology 2)domain protein that can transform fibroblasts and differentiate PC12cells in a RAS-dependent fashion. Shc binds a variety of tyrosinephosphorylated growth factor receptors presumably via itscarboxyl-terminal SH2 domain. We cloned a fragment of Shc when screeninga bacterial expression library with tyrosine-phosphorylated epidermalgrowth factor (EGF) (epidermal growth factor) receptor. Surprisingly,this fragment encodes the amino terminus of Shc, a region that has nosignificant similarity to an SH2 domain. When expressed as a glutathioneS-transferase fusion protein, this amino-terminal domain binds toautophosphorylated EGF receptor, as well as HER2/neu and TrkA receptors.This fragment acts like an SH2 domain in that it does not bindnon-phosphorylated EGF receptor or EGF receptor with all tyrosinephosphorylation sites mutated or deleted. Our data define a novel domainin Shc that has the potential to interact with growth factor receptorsand other tyrosine-phosphorylated proteins.

Growth factor receptors with intrinsic tyrosine kinase activity undergoautophosphorylation on multiple tyrosine residues upon binding ligand.These tyrosine autophosphorylation sites then serve as binding sites forSH2 domain proteins (Fantl, W. J., et al., (1993) Annu. Rev. Biochem.62, 453-481). Genetic and biochemical evidence demonstrates that SH2domains have a crucial role in the response of cells to growth factors(Pawson, T. and Schlessinger, J. (1993) Curr. Biol. 3, 434-442; andMayer, B. J. and Baltimore, D. (1993) Trends Cell Biol. 3, 8-13). Ourlaboratory has been cloning SH2 domain proteins based on their abilityto bind to the tyrosine-phosphorylated EGF receptor (Skolnik, E. Y., etal. (1991) Cell 65, 83-90; Lowenstein, E. J., et al. (1992) Cell 70,431-442; and Margolis, B., et al. (1992) Proc. Natl. Acad. Sci. USA 89,8894-8898). This method involves screening bacterial expressionlibraries with the radioactively labeled carboxyl-terminal tail of theEGF receptor. We have termed the method CORT (Cloning Of ReceptorTargets) and the proteins isolated GRBs (growth Factor Receptor Bound).One of the genes cloned in this manner, Grb2, has homologues inCaenorhabditis elegans and Drosophila melanogaster and is crucial forviability and development (Schlessinger, J. (1993) Trends Biochem. Sci.18, 273-275). Grb2 binds to a second protein, Son of Sevenless (Sos),which acts as a GDP/GTP exchanger for Ras. In this manner Grb2 acts asan adapter protein coupling growth factor receptor to Ras. Another genecloned by this method is Grb7, an SH2 domain protein of unknown functionthat binds to EGF receptor and is overexpressed in breast cancer (Stein,D., et al. (1994) EMBO J. 13, 1331-1340).

SH2 domain proteins such as Grb2 and Grb7 bind not only totyrosine-phosphorylated growth factor receptors but also totyrosine-phosphorylated cytoplasmic proteins. For example, the SH2domain protein Shc becomes tyrosine-phosphorylated after EGF receptoractivation and binds Grb2 and Grb7 (Stein, D., et al. (1994) EMBO J. 13,1331-1340; Pelicci, G., et al. (1992) Cell 70, 93-104; andRozakis-Adcock, M., et al. (1992) Nature 360, 689-692). Shc is expressedas three different proteins of 46, 52 and 66 kDa. The p46^(Shc) andp52^(Shc) forms arise by alternate translational start sites while thep66^(Shc) form is generated by alternate splicing (Pelicci, G., et al.(1992) Cell 70, 93-104). Shc tyrosine phosphorylation is induced aftercell activation with a variety of growth factors and cytokines (Pelicci,G., et al. (1992) Cell 70, 93-104; and Cutler, R. L., et al. (1993) J.Biol. Chem. 268, 21463-21465). It is also phosphorylated in cellsexpressing activated non-receptor tyrosine kinases such as v-src(McGlade, J., et al. (1992) Proc. Natl. Acad. Sci. USA 89, 8869-8873).Shc overexpression results in transformation of fibroblasts anddifferentiation of PC12 cells. The differentiation of PC12 cells isblocked by dominant interfering mutations of Ras consistent with therole of Shc to bind the Grb2-Sos complex (Rozakis-Adcock, M., et al.(1992) Nature 360, 689-692).

The interaction of Shc with tyrosine-phosphorylated growth factorreceptors such as EGF receptor, TrkA and HER2/neu as well as othertyrosine-phosphorylated proteins such as middle T antigen is wellestablished (Pelicci, G., et al. (1992) Cell 70, 93-104; Segatto, O., etal. (1993) Oncogene 8, 2105-2112; Obermeier, A., et al. (1994) EMBO J.13, 1585-1590; and Stephens, R. M., et al. (1994) Nature 367, 87-90).Shc appears to interact with tyrosine-phosphorylated proteins bearingthe sequence, Asn-Pro-X-Tyr (P). This interaction is unusual for SH2domains that usually select specificity based on amino acidscarboxyl-terminal to the phosphotyrosine (Songyang, Z., et al. (1994)Mol. Cell. Biol. 14, 2777-2785). Nonetheless, studies have indicatedthat the specificity of Shc binding to tyrosine-phosphorylated proteinsis determined by the SH2 domain (Batzer, A. G., et al. (1994) Mol. Cell.Biol. 14, 5192-5201; and Okabayashi, Y., et al. (1994) J. Biol. Chem.269, 18674-18678). In this report we demonstrate that Shc has a seconddomain in its amino terminus, distinct from the SH2 domain, that caninteract with tyrosine-phosphorylated growth factor receptors. The datasuggest a novel mechanism whereby signaling molecules can interact withgrowth factor receptors.

Materials and Methods

Library Screening and Cloning of Murine Shc—A randomly primed λEXloxlibrary was prepared from NIH 3T3 cells. Approximately 1 million phageswere screened using the tyrosine-phosphorylated carboxyl terminus of theEGF receptor as previously described (Skolnik, E. Y., et al. (1991) Cell65, 83-90; Lowenstein, E. J., et al. (1992) Cell 70, 431-442; andMargolis, B., et al. (1992) Proc. Natl. Acad. Sci. USA 89, 8894-8898).Nine clones were obtained and will be presented in detail elsewhere. Thepositive phages were purified, isolated, converted to plasmids, andsequenced as previously described (Margolis, B., et al. (1992) Proc.Natl. Acad. Sci. USA 89, 8894-8898). The original insert encoding thefirst 209 amino acids of p52^(Shc) was then used as a DNA probe toisolate a full-length Shc cDNA from a 16 day embryonic mouse library(Novagen, Madison, Wis.).

DNA Constructs—Using murine p52 ^(Shc) cDNA as a template, DNAcorresponding to the areas of interest were synthesized using polymerasechain reaction with oligonucleotides that contained EcoRI restrictionsites to allow for subcloning. The amplified DNA was isolated, digestedwith EcoRI and subcloned into the pGSTag expression vector (Ron, D. andDressler, H. (1992) Biotechniques 13, 866-869). Deletion mutants werealso generated by polymerase chain reaction using standard techniques(Ausubel, F. M., et al. Current Protocols in Molecular Biology, (1992)Wiley Interscience, New York). All constructs were sequenced usingSequenase Version 2.0 (U.S. Biochemical Corp.).

Cell Culture—HER 14 cells (NIH 3T3 cells expressing ˜4×10⁵ human EGFreceptors/cell), NIH 3T3 cells overexpressing a chimeric receptor thatlinks the extracellular domain of the EGF receptor to the intracellulardomain of the HER2/neu (HER 1/2), and NIH 3T3 cells expressingautophosphorylation mutants of the EGF receptor have been describedelsewhere (Lee, J., et al. (1989) EMBO J. 8, 167-173; Honegger, A. M.,et al. (1987) Cell 51, 199-209; and Li, N., et al. (1994) Oncogene (InPress)). These cells were grown in Dulbecco's modified Eagle's mediumwith 10% calf serum with 50 U/ml penicillin and 50 μg/ml streptomycin.PC12 cells were grown in Dulbecco's modified Eagle's medium with 10%horse serum, 5% fetal calf serum, and penicillin/streptomycin. NIH 3T3cells were starved overnight in 1% calf serum and then stimulated withEGF for 3 min. PC12 cells were starved for 48 hours in 1% fetal calfserum and 1% horse serum and then stimulated with nerve growth factorfor five min. Cells were lysed in 1% Triton X-100 lysis buffercontaining protease and phosphatase inhibitors (Margolis, B., et al.(1989) Cell 57, 1101-1107).

Antibodies—To analyze EGF receptor, mAb (monoclonal antibody) 108 wasused for immunoprecipitation while antipeptide antibodies, anti-C andanti-F, were used for immunoblotting as previously described (Margolis,B., et al. (1990) EMBO J. 9, 4375-4380). Rabbit polyclonal TrkAantibodies were purchased from Oncogene Science (Uniondale, N.Y).Polyclonal GST (glutathione S-transferase) antibodies were prepared byinjecting rabbits with purified GST protein and anti-phosphotyrosineantibodies were prepared as previously described (Margolis, B., et al.(1990) EMBO J. 9, 4375-4380).

Binding Studies—GST fusion proteins were expressed using standardtechniques (Ausubel, F. M., et al. Current Protocols in MolecularBiology, (1992) Wiley Interscience, New York). Protein concentration wasdetermined by SDS-PAGE (polyacrylamide gel electrophoresis) using bovineserum albumin as a standard. Lysates were incubated with GST fusionproteins bound to glutathione-agarose beads for 90 minutes at 4° C. Thebeads were then washed three times with HNTG (20 mM Hepes, pH 7.5, 150mM NaCl, 10% glycerol, and 0.1% Triton X-100), boiled in 1× samplebuffer, and separated by SDS-PAGE. Transfer and immunoblotting wereperformed as described (Margolis, B., et al. (1989) Cell 57, 1101-1107).In other studies, EGF receptor was immunoprecipitated with mAb 108 andwashed three times with HNTG. Receptors were then phosphorylated invitro using 5 mM MnCl₂ and 50 μM ATP in HNTG. After washing three moretimes with HNTG, 1 ml of 1% Triton X-100 lysis buffer containing 5 μg ofGST fusion protein as well as protease and phosphatase inhibitors wasadded to the immobilized receptors. After incubating for 90 minutes at4° C., samples were washed three more times with HNTG and boiled in 1×sample buffer. Forty microliters of post binding supernatant and thebound proteins were separated by SDS-PAGE, transferred tonitrocellulose, and blotted with anti-GST.

Results and Discussion

In an effort to isolate new Grb proteins we generated a randomly primedbacterial expression library from NIH 3T3 cells. One clone, Grb12, boundto the probe with a similar intensity as that seen with other clonesisolated in this screen such as one encoding the SH2 domain of Grb2(FIG. 1A). Surprisingly, Grb12 did not contain an SH2 domain but ratherencoded 209 amino acids of murine Shc identical to the amino terminus ofhuman p52^(Shc). We proceeded to clone the cDNA that encoded thefull-length murine p52^(Shc) and found it to be 96% identical to humanShc (FIG. 1B).

These results were interesting because they suggested that Shc mightinteract with growth factor receptors in a novel fashion. Accordingly weexamined the binding of the Shc amino terminus (hereafter referred to asShc 1-209) in further detail. First we determined if the binding of Shc1-209 was dependent on EGF receptor activation. To perform these studieswe generated Shc 1-209 as a GST fusion protein and examined its abilityto bind EGF receptor from lysates of NIH 3T3 cells. We found that, likethe Grb7SH2 domains, Shc 1-209 bound only to the activatedtyrosine-phosphorylated receptor and not to the inactivenon-phosphorylated receptor (FIG. 2A). We asked if this binding wasdependent on receptor autophosphorylation. EGF receptor wasimmunoprecipitated from unstimulated cells and thentyrosine-phosphorylated in vitro by adding Mn²⁺ and ATP. We then addedbacterial lysates expressing Shc 1-209 and found that Shc 1-209 bounddirectly to phosphorylated but not non-phosphorylated receptors (FIG.2B). To confirm that the binding was dependent on autophosphorylation,we examined the binding of Shc 1-209 to EGF receptor mutants lackingautophosphorylation sites. We found that mutation of all EGF receptortyrosine-phosphorylated residues to phenylalanine or deletion of the EGFreceptor carboxyl terminus completely eliminated Shc 1-209 binding (FIG.3). It was found that while 30 mM phosphotyrosine could partiallyinhibit the binding of Shc 1-209 to the EGF receptor, it completelyinhibited the binding of the Grb7 SH2 domain to this receptor (resultsnot shown). This may represent a difference in the affinity ofinteractions or a basic difference in the molecular mechanism of EGFreceptor interaction with Shc 1-209 versus SH2 domains.

The binding of Shc 1-209 was compared to the binding of the Shc SH2domain to EGF receptor. Consistently, we found that the EGF receptorbinding of Shc 1-209 was comparable or superior to that observed withthe Shc SH2 domain (FIG. 4B). This was found not only with the murineShc SH2 domain, but also with the human Shc SH2 domain (results notshown). Furthermore we found that Shc 1-209 bound to HER2/neu and TrkA(FIGS. 4, C and D). In the case of TrkA, Shc 1-209 appeared to bind in asimilar fashion to that seen with the SH2 domain of phospholipase C-λ.

p46^(Shc) is encoded by a protein that begins 46 amino acidscarboxyl-terminal to the start site for p52. We asked if the amino acidsencoding the amino terminus of p46^(Shc), i.e. Shc 46-209, could alsobind to growth factor receptors. Our results indicate that this domainas present in p46^(Shc) can bind both EGF receptor and HER2/neureceptor. However a construct with a more severe amino-terminaldeletion, Shc 85-209, did not bind growth factor receptors nor did aconstruct Shc 1-209Δ106-117 with amino acids 106-117 deleted (FIG.4A-C).

In summary, our results reveal a novel growth factor receptor bindingdomain in the amino terminus of Shc. The binding to the EGF receptorappears direct and tyrosine phosphorylation dependent in a fashionsimilar to that seen with the SH2 domain. Although the domain behaveslike an SH2 domain, sequence analysis of this domain reveals nosignificant similarity to SH2 domain proteins or any other protein inthe data base. The binding is not dependent on fusion of this domain toGST as binding was also seen in the library screening where Shc 1-209was fused to gene 10 of the T7 phage. This result appears to reveal anew mechanism whereby proteins can interact with growth factor receptorsand other tyrosine-phosphorylated proteins.

One however must approach the data presented here with some caution. Allour studies are based on in vitro interactions with fusion proteins thatmay not be representative of what is seen with the full-length Shcprotein. We have just begun to study the effect of SH2 domain andamino-terminal domain mutations on the binding of full-length Shcexpressed in mammalian cells. Mutations in the highly conserved FLVRsequence in the SH2 domain appear to reduce the binding of Shc to EGFreceptor by approximately 90%. In contrast, deletions in theamino-terminal domain (such as the removal of the amino-terminal 85amino acids) reduce the binding of Shc to EGF receptor by only 50%. Thusthese preliminary observations suggest the amino-terminal domain cancooperate with the SH2 domain to promote binding to growth factorreceptors. This might be akin to the cooperative effect seen when thetwo SH2 domains of phosphatidylinositol 3 kinase associated p85 bind tothe platelet-derived growth factor receptor (Kashishian, A., et al.(1992) EMBO J. 11, 1373-1382). It might also explain the highstoichiometry of association between Shc and EGF receptor (Soler, C., etal. (1994) J. Biol. Chem. 269, 12320-12324). It is not entirely clearwhy the amino terminus cannot completely compensate for reductions inthe SH2 domain binding when one considers the strength of binding wehave observed with GST fusion proteins or in the library screening.Without knowledge of the structure of the Shc protein it is difficult toknow whether the amino terminus of Shc is always surface exposed andable to play a role in protein-protein interactions. The binding of theamino-terminal domain to proteins may also be affected by reversiblemodification such as phosphorylation. It is also possible that theamino-terminal domain may play an important role in binding only aspecific subset of tyrosine-phosphorylated proteins.

The role of the amino-terminal domain in the physiologic interaction ofShc with tyrosine-phosphorylated proteins is unknown. Yet theobservation that the amino terminus of Shc when expressed alone bindstightly to several different growth factor receptors is striking. It ispossible that the amino terminus of Shc might in some fashion contributeto the unusual ability of Shc to bind the Asn-Pro-X-Tyr (P) motif. It isclear that there is much more to learn about the molecular basis of Shcfunction and its interaction with growth factor receptors. Our findingsindicate that there may be a heretofore unappreciated mechanism wherebysignaling molecules can interact in a specific fashion withtyrosine-phosphorylated proteins. Further studies on the function of theShc amino-terminal domain should provide insight into this problem.

Other embodiments are within the following claims.

1-12. (Canceled)
 13. A method of assaying for agents useful fordisrupting APB interactions or in the diagnosis of APB diseases ordisorders, comprising the step of measuring the ability of an agent tobind to an APB recognition region or an APB domain.
 14. The method ofclaim 13, wherein said APB domain has an amino acid region with at least30% sequence similarity to the Shc APB domain.
 15. The method of claim13, wherein said APB domain has an amino acid region with at least 20%sequence identity to the Shc APB domain. 16-18. (Canceled)
 19. Themethod of claim 13, wherein the ability of an agent to bind to an APBrecognition region is measured.
 20. The method of claim 13, wherein theability of an agent to bind to an APB domain is measured.
 21. The methodof claim 13, wherein said agent is an antibody.
 22. The method of claim13, wherein said agent is a polypeptide comprising at least 10 aminoacids, including the amino acid sequenceAsparagine-Proline-X-(phosphorylated) Tyrosine.
 23. A method ofidentifying an agent able to bind to an APB domain or an APB recognitionregion, comprising: (a) exposing at least one agent to a proteincomprising an APB domain or an APB recognition region for a timesufficient to allow binding of the agent; (b) removing non-bound agents;and (c) determining the presence of bound agent.
 24. The method of claim23, wherein multiple agents are simultaneously screened.
 25. A method ofidentifying an agent able to promote APB binding complexes, comprising:(a) exposing a first polypeptide containing an APB domain or APBrecognition region to a candidate agent; and (b) measuring the abilityof a second polypeptide containing an APB domain or APB recognitionregion to form a complex with said first polypeptide containing an APBdomain or APB recognition region.
 26. A method of identifying an agentable to promote APB binding complexes, comprising: (a) exposing a cellcapable of forming an APB binding complex to a candidate agent; (b)lysing said cell; (c) exposing the cell lysate to an antibody that bindsto a component of an APB binding complex; and (d) measuring APB complexformation.
 27. The method of claim 26, wherein step (d) employsimmunoprecipitation.
 28. The method of claim 26, wherein said antibodyis bound to a solid support.